Analysis of Denosumab by a Validated CZE Method and Determination of Sialic Acids by the RP-HPLC Method.

A capillary zone electrophoresis (CZE) method was developed and validated to quantitate the monoclonal antibody denosumab (DmAb) and its charge variants in pharmaceutical products, demonstrating excellent precision, linearity and accuracy. Separations were obtained with migration times of 11.3 min for DmAb and the calibration curve was linear in the range of 0.95-20 mg/mL. The analytical comparability of seven batches of Prolia® showed mean differences of the estimated content/potencies of 1.87% lower, and 0.84 and 1.21% higher compared with the size-exclusion and reversed-phase liquid chromatography (SE-HPLC and RP-HPLC) methods and the osteoclast antiproliferative bioassay, respectively, with non-significant differences (P > 0.05). An RP-HPLC method with fluorescence detection (RP-HPLC-F), performed on a Kinetex® EVO C18 column (5 µm, 100 Å, 250 mm × 4.6 mm), was optimized to determine the levels of sialic acids of DmAb biomolecules, giving mean concentrations of 0.16 and 0.17 µg N-acetylneuraminic acid/mg DmAb for Prolia® and Xgeva® pharmaceutical products, respectively. The results demonstrated the capability of each one of the methods, and their use in combination constitutes a strategy to monitor instability, thereby assuring the quality and the batch-to-batch consistency of the biotechnology-derived medicine.

Quantitation of certolizumab pegol by validated liquid chromatography methods

Abstract Certolizumab pegol (CZP) is a Fab' fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn's disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.